1. Technical Field
The present invention relates to the effects of the administration of hydrogen and the increase of hydrogen in the body with regard to health promoting benefits. In particular, the present invention relates to effects of an aqueous solution including hydrogen suppressing the biological cell-signaling mechanism that causes inflammation.
2. Background Art
It has previously been reported that molecular hydrogen (H2) dissolved in water, due to its antioxidant effects, has therapeutic value in the mouse model of brain injury induced by ischemia reperfusion. More specifically, the ability of H2 to reduce the reactive oxygen species (ROS) generated in the course of brain injury was revealed to be responsible for this H2-mediated therapeutic effect. Following this study, several other reports demonstrated that H2 could suppress tissue injury in organs, such as liver, intestine, kidney and heart, caused by oxidative stress following ischemia reperfusion or transplantation induced graft injury. In addition, recently, Applicants revealed that H2 can also exert anti-inflammatory effects by demonstrating the H2-mediated protective effects in DSS-induced colitis model and cocanavalin A-induced hepatitis. However, since the biological processes of inflammation and oxidative stress are closely associated, the precise mechanism whereby H2 prevents inflammatory response has remained unclear.
Mitogen-activated protein kinase (MAPK) signal transduction pathways play a key role in inflammatory cell signaling. MAPK pathways can be activated by a wide variety of inflammatory stimuli including one mediated by bacterial endotoxin, lipopolysaccharide (LPS), and different stress acting through diverse receptor families, including hormone receptors, growth factor receptors or cytokine receptors, seven-transmembrane receptors and through environmental stresses such as osmotic shock, ionizing radiation and ischemic injury. MAPK pathways, in turn, coordinate activation of gene transcription, protein synthesis, cell cycle machinery, cell death, and differentiation, in the context of inflammatory and stress responses.
Mammalian MAP kinases can be divided into three groups based on their structure and function: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase or stress activated protein kinase (JNK or SAPK), ERK5 or BMK, and p38 group. A variety of extracellular stimuli produce cellular responses via activation of the MAP kinase cascades. To date three MAP kinase pathways, ERK, JNK, and p38, have been reported to be activated by LPS stimulation in macrophages.
ERK is the first group of MAP kinases that was found to be activated by LPS stimulation. In response to LPS stimulation, two tyrosine phosphorylated MAP kinase proteins, p42 (ERK2) and p44 (ERK1), can be detected in macrophages. Activation of ERKs can occur via the upstream effector, Ras. Upon stimulation, Ras interacts with the NH2-terminal domain of Raf-1, leading to its recruitment to the plasma-membrane, whereupon Raf-1 is phosphorylated by another kinase. Once activated, Raf-1 can phosphorylate MAP kinase kinase-1 (MKK-1, also known as MEK), which in turn, phosphorylates ERK.
Upon exposure to LPS, ERK-½ and its upstream activator, MEK-1 has been shown to be activated in monocytes, which elicits production of TNF-α or IL-1b in response to LPS treatment, indicating that ERKs may be involved in the signaling pathway that results in cytokine synthesis following LPS treatment. The Ras and Raf-1 components of the ERK cascade have been suggested to be necessary for LPS-stimulated TNF-α production. This was shown from the ability of LPS to induce rapid phosphorylation of Raf-1 in macrophages, activating the MEK-1/ERK pathway. However, while up-regulation of Raf-1 has been shown to strongly activate ERK1 and ERK2, it causes only a small increase in TNF-α mRNA expression, and protein secretion. Thus, the ERK pathway only partially mimics LPS effects, implying that other signaling pathways triggered by LPS may also play a role. Indeed, it has been demonstrated that LPS stimulation leads to the activation of multiple signal pathways, including JNK, and p38 MAP kinase pathways.
Both JNK1 and JNK2 have been reported to be activated in LPS treated macrophages. As their name indicates, the JNK proteins are associated with the phosphorylation of c-Jun. In addition, other transcription factors, including activating transcription factor-2 (ATF-2), and ternary complex factor (TCF) have been reported to be downstream targets for JNK. c-Jun can complex with c-fos or ATF2 which constitutes AP-1 or CRE binding activity. TCF mediated c-fos expression also influences c-Jun/c-fos dimer formation which would have impact on AP-1 binding activity. Since AP-1 and CRE sites are found in many cytokine promoters including TNF-a, regulation of AP-1 and/or CRE binding activity by JNK pathway plays important role in LPS induced cytokine expression. In addition, c-Jun complexes have been shown to act synergistically with NF-k B in LPS-treated monocytes to enhance the induction of TNF-α.
p38 (or p38a, also known as CSBP and RK) was initially isolated and cloned through a study designed to identify proteins in macrophages and pre-B cells that tyrosine phosphorylated in response to LPS. p38a was also cloned as a specific target of pyridinyl imidazole derivatives such as SB203580 which inhibit the production of proinflammatory cytokines by monocytes. Four isoforms of the p38 MAP kinases have been cloned and characterized: p38 (p38a), p38b, p38gamma (also known as ERK6 or SAPK3), and p38delta (also known as SAPK4), each of which contains a TGY dual phosphorylation motif between domains VII and VIII—distinguishing them from the ERK kinases (TEY) and JNK kinases (TPY). p38a and p38b are sensitive to SB203580 inhibition, but the activity of p38gamma and p38delta are unaffected. The discovery of a specific inhibitor of p38 SB203580 has provided a useful tool for dissecting the role of p38 kinases in septic shock, while its application to human patients has not been approved by FDA. Studies have demonstrated that inhibition of p38 in monocytes prevents LPS-stimulated production of IL-1b and TNF. It is clear that p38 pathways play a crucial role in LPS induced cytokine expression. However, the precise mechanism by which p38 regulates cytokine gene expression is still uncertain.
The MAPK signal transduction pathway also plays a central role in regulating tumor cell growth, survival, differentiation, and angiogenesis. The key components of the MAPK signal module (Ras/Raf/MEK/ERK) are frequently altered in human cancers. Targeting this pathway represents a promising anticancer strategy. Mutations in the small molecular weight G-proteins “Ras family” of proto-oncogenes are very common, being found in 20% to 30% of all human tumors. Therefore, Raf-MEK-ERK MAPK pathway represents one of the best characterized Ras signaling pathways which play a key role in growth of certain types of cancer cells. Raf and MEK have consequently emerged as key protein kinases to target for anticancer drug design. As noted above, while there exist multiple MAP kinase families, e.g. c-Jun N-terminal kinase (JNK) and p38, which are also activated downstream of small molecular weight G-proteins, ERK has been the best characterized and is more pertinent to aberrant signaling in human cancer. For some cancers, especially those of hematopoietic origin, the p38 and JNK pathways may in fact yield targets exploitable for anticancer drug development. However, a broad array of solid tumors is known to express constitutive levels of phosphorylated ERK1 and ERK2. Activation of ERK is critical for a large number of Ras-induced cancer cell responses.
MAP kinase phosphatases (MKPs) are a class of molecule that suppresses MAP kinase activity. MKPs are dual-specificity phosphatases (DUSPs) that recognize the TXY (Threonine: X-amino acid: Tyrosine) amino acid motif present in MAPK family members. More specifically, MKPs are uniquely able to hydrolyze the phosphate ester bond on both a tyrosine (Y) and a threonine (T) residue on the same protein. MKP expression can be induced by factors that activate MAPKs, such as environmental stresses and growth factor stimulation. Among a total of 11 MKP family members, the most frequently studied member of the MKP family is MKP-1. MKP-1 can dephosphorylate all three members of the MAPK family, but it has a much higher affinity for JNK and p38, with a much lower affinity for ERK. Given the pivotal role of MKP-1 in innate immunity, there is growing interest in exploring how its gene expression and biochemical activity are regulated. Expression of MKP-1 is induced by a number of growth factors and stresses in multiple cell types. In macrophages responding to activation of TLR by its ligand, such as LPS, there is a strong and rapid induction of MKP-1 mRNA and increase in MKP-1 protein abundance, peaking at 1 hour after stimulation. The induction of MKP-1 correlates with a decline in the activities of JNK and p38 MAPK, which is consistent with a role for MKP-1 in the inactivation of these MAPKs as a feedback mechanism to restrain excessive inflammation.
Therefore, there is a need for a composition that can modulate the level of MAPK at the cellular level in order to reduce and control inflammation.